Driving Biomedical Projects (DBPs) Summary We selected the Driving Biomedical Projects (DBPs) for this proposal to encompass the forefront in biomedical research and cover the US from coast to coast. Some of the most fundamental questions in biochemistry are being addressed using microcrystal electron diffraction (MicroED): How do ligands regulate the structure and function of G-protein coupled receptors? (Lefkowitz, Stevens); What are the conformational dynamics that are involved in substrate recognition and transport in the largest family of membrane transporters? (Abramson, Zheng); Structural biology of large porous protein complexes that are involved in neurotransmission and fusion (Hurley); The protonation and charged state in membrane proteins and how those affect protein function (Rees, Yeager); How proteins are recognized by antibodies and how proteins can regulate gene expression (Bjorkman, Feigon); and the utility of MicroED in understanding and discovery of natural products (van der Donk). We chose to focus our efforts on the study of natural products, protein complexes and of membrane proteins because these are extremely challenging and yet very important targets for drug discovery and medicine. The interactions with the DBPs will be supervised by Dr. Gonen, who has more than 20 years' experience working with membrane proteins of different kinds, including, channels, transporters and receptors and has determined several key membrane protein structures by cryoEM and X-ray crystallography. Moreover, he holds a degree in Chemistry which allows him to effectively communicate with chemists and biologists alike. Many of the DBP targets are hard to express, purify and crystallize. By applying MicroED we help to alleviate these difficulties; when a membrane protein of interest if expressed in low amounts, the resulting material may still be sufficient for structure determination by MicroED. Finally, MicroED can deliver atomic resolution structures from femtogram amounts of small molecules and natural products without additional purification and crystallization . All DBPs benefit from the Technology and Research Development projects of this application and are good testbeds for them. They are also excellent vehicles for dissemination of MicroED technology.